Molecular population genetics of Cucumber mosaic virus in California: evidence for founder effects and reassortment

The structure and genetic diversity of a California Cucumber mosaic virus (CMV) population was assessed by single-strand conformation polymorphism and nucleotide sequence analyses of genomic regions 2b, CP, MP, and the 3' nontranslated region of RNA3. The California CMV population exhibited low genetic diversity and was composed of one to three predominant haplotypes and a large number of minor haplotypes for specific genomic regions. Extremely low diversity and close evolutionary relationships among isolates in a subpopulation suggested that founder effects might play a role in shaping the genetic structure. Phylogenetic analysis indicated a naturally occurring reassortant between subgroup IA and IB isolates and potential reassortants between subgroup IA isolates, suggesting that genetic exchange by reassortment contributed to the evolution of the California CMV population. Analysis of various population genetics parameters and distribution of synonymous and nonsynonymous mutations revealed that different coding regions and even different parts of coding regions were under different evolutionary constraints, including a short region of the 2b gene for which evidence suggests possible positive selection.     

Biological and Molecular Characterization of Taiwanese Isolates of Cucumber mosaic virus Associated with Banana Mosaic Disease

Banana mosaic disease (BMD) caused by Cucumber mosaic virus (CMV) has become an important threat to the banana industry. We collected and characterized 10 CMV isolates associated with BMD in Taiwan and compared their biological characteristics and coat protein sequences. The isolates fell into four pathotypes on the basis of the symptoms they induce on banana, Nicotiana glutinosa and Vigna unguiculata (cowpea). Double-stranded RNA analysis revealed that the different pathotypes are not related to the presence of CMV satellite RNA. Phylogenetic analysis of worldwide CMV coat protein sequences revealed that among the currently known CMV subgroups IA, IB and II, subgroup IB is phylogenetically unresolved. Our CMV isolates form a new subgroup, IT, within subgroup I. In addition, we resolved another new CMV subgroup, IS, within subgroup I. The analysis also revealed that isolates within different subgroups can infect the banana.     

Rearrangements in the 5′ Nontranslated Region and Phylogenetic Analyses of Cucumber Mosaic Virus RNA 3 Indicate Radial Evolution of Three Subgroups

Cucumber mosaic virus (CMV) has been divided into two subgroups based on serological data, peptide mapping of the coat protein, nucleic acid hybridization, and nucleotide sequence similarity. Analyses of a number of recently isolated strains suggest a further division of the subgroup I strains. Alignment of the 5' nontranslated regions of RNA 3 for 26 strains of CMV suggests the division of CMV into subgroups IA, IB, and II and suggests that rearrangements, deletions, and insertions in this region may have been the precursors of the subsequent radiation of each subgroup. Phylogeny analyses of CMV using the coat protein open reading frame of 53 strains strongly support the further division of subgroup I into IA and IB. In addition, strains within each subgroup radiate from a single point of origin, indicating that they have evolved from a single common ancestor for each subgroup.     

Evolutionary history of Cucumber mosaic virus deduced by phylogenetic analyses

Cucumber mosaic virus (CMV) is an RNA plant virus with a tripartite genome and an extremely broad host range. Previous evolutionary analyses with the coat protein (CP) and 5' nontranslated region (NTR) of RNA 3 suggested subdivision of the virus into three groups, subgroups IA, IB, and II. In this study 15 strains of CMV whose nucleotide sequences have been determined were used for a complete phylogenetic analysis of the virus. The trees estimated for open reading frames (ORFs) located on the different RNAs were not congruent and did not completely support the subgrouping indicated by the CP ORF, indicating that different RNAs had independent evolutionary histories. This is consistent with a reassortment mechanism playing an important role in the evolution of the virus. The evolutionary trees of the 1a and 3a ORFs were more compact and displayed more branching than did those of the 2a and CP ORFs. This may reflect more rigid host-interactive constraints exerted on the 1a and 3a ORFs. In addition, analysis of the 3' NTR that is conserved among all RNAs indicated that evolutionary constraints on this region are specific to the RNA component rather than the virus isolate. This indicates that functions other than replication are encoded in the 3' NTR. Reassortment may have led to the genetic diversity found among CMV strains and contributed to its enormous evolutionary success.     

Reappearance of Cucumber mosaic virus Isolates Belonging to Subgroup IB in Tomato Plants in North-eastern Spain

A disease characterized by symptomless leaves and fruit decolouration, loss of consistency and mild deformation on ripening was detected in tomato fields in north‐eastern Spain during 2003 and 2004. DAS‐ELISA analysis revealed the presence of the Cucumber mosaic virus (CMV) in all diseased plants. CMV isolates were characterized by polyacrylamide gel electrophoresis (PAGE) analysis of double‐stranded RNAs (dsRNAs) and nucleotide sequence analysis, and compared with some CMV isolates belonging to different subgroups used as controls. A total of 12 isolates obtained from the infected tomato plants selected for this study gave the same electrophoresis pattern for the three genomic dsRNAs, which was different to the patterns showed by the CMV isolates collected in the same region a few years ago. The identity of the complete nucleotide sequence of one of these CMV isolates and the partial sequence of other five isolates compared to the Tfn strain from Italy and the BAR92/1 isolate from tomato collected in Barcelona in 1992 was higher than 99%, both belonging to subgroup IB of CMV. The CMV isolates of this subgroup found in eastern Spain in previous studies were not detected after 1996. The nucleotide sequences of two isolates that were chosen as representatives of the CMV isolates more frequently detected in previous years revealed that they belonged to the CMV subgroups IA. The origin and the possible causes of reappearance of CMV IB isolates in north‐eastern Spain are discussed.     

Characterization, Genetic Diversity, and Evolutionary Link of Cucumber mosaic virus Strain New Delhi from India

The genome of Cucumber mosaic virus New Delhi strain (CMV-ND) from India, obtained from tomato, was completely sequenced and compared with full genome sequences of 14 known CMV strains from subgroups I and II, for their genetic diversity. Sequence analysis suggests CMV-ND shares maximum sequence identity at the nucleotide level with a CMV strain from Taiwan. Among all 15 strains of CMV, the encoded protein 2b is least conserved, whereas the coat protein (CP) is most conserved. Sequence identity values and phylogram results indicate that CMV-ND belongs to subgroup I. Based on the recombination detection program result, it appears that CMV is prone to recombination, and different RNA components of CMV-ND have evolved differently. Recombinational analysis of all 15 CMV strains detected maximum recombination breakpoints in RNA2; CP showed the least recombination sites.     

Genetic Diversity Among Iranian Isolates of Rhizoctonia solani Kühn Anastomosis Group1 Subgroups Based on Isozyme Analysis and Total Soluble Protein Pattern

Rhizoctonia solani is a destructive fungal pathogen with a wide host range. The R. solani complex species includes several divergent groups delimited by affinities for hyphal anastomosis. In this study, genetic variation among 20 isolates of R. solani anastomosis group 1 (AG1) subgroups (AG1‐IA and AG1‐IB) collected from Mâzandaran province, Iran, and standard isolates of these subgroups, was determined by isozyme analysis and total soluble protein profile. Mycelial protein pattern and isozyme analysis were studied using denaturing and non‐denaturing polyacrylamide gel electrophoresis, respectively. A total of 15 enzyme systems were tested, among which six enzymes including esterase, alkaline phosphatase, superoxide dismutase, octanol dehydrogenase, lactate dehydrogenase and mannitol dehydrogenase generated distinct and reproducible results. The soluble protein patterns were similar among the R. solani isolates examined; however, minor differences in banding pattern were observed between the two subgroups. In isozyme analysis, a total of 64 electrophoretic phenotypes were detected for all six enzymes used. Based on cluster analysis and similarity matrix, the fungal isolates were divided into two genetically distinct groups of I and II consistent with the previously reported AG1‐IA and AG1‐IB subgroups in AG1. Group I represented all isolates belonging to AG1‐IA subgroup, whereas group II represented all isolates belonging to AG1‐IB subgroup. Results from isozyme analysis suggest that the subgrouping concept within AGs is genetically based.     

Naturally occurring isolates of Neisseria gonorrhoea, which display anomalous serovar properties, express PIA/PIB hybrid porins, deletions in PIB or novel PIA molecules

The por gene of Neisseria gonorrhoeae encodes the Protein I porin responsible for serovar specificity. In this study the por genes have been sequenced from clinical isolates which exhibited anomalous serovar reactivity. One group of `intermediate' strains differed significantly from both Protein IA and IB strains, were more closely related to IA but appeared to represent a distinct class of Protein I. Another strain was closely related to Protein IB of serovar IB-6 but contained a deletion of six amino acids in surface exposed loop 6 which removed epitopes recognised by IB specific monoclonal antibodies. The third group of strains, which reacted with both IA and IB specific monoclonal antibodies, expressed hybrid Protein I molecules containing both IA and IB epitopes. These strains appeared to originate from a double crossover between Proteins IA and IB with the amino and carboxy terminal residues homologous to IB while the surface exposed loop 6 demonstrated close homology to IA. This is the first demonstration of naturally occurring gonococci expressing a hybrid Protein IA/IB.     

Widespread Recombination,Reassortment, and Transmission of Unbalanced Compound Viral Genotypes in Natural Arenavirus Infections

Arenaviruses are one of the largest families of human hemorrhagic fever viruses and are known to infect both mammals and snakes. Arenaviruses package a large (L) and small (S) genome segment in their virions. For segmented RNA viruses like these, novel genotypes can be generated through mutation, recombination, and reassortment. Although it is believed that an ancient recombination event led to the emergence of a new lineage of mammalian arenaviruses, neither recombination nor reassortment has been definitively documented in natural arenavirus infections. Here, we used metagenomic sequencing to survey the viral diversity present in captive arenavirus-infected snakes. From 48 infected animals, we determined the complete or near complete sequence of 210 genome segments that grouped into 23 L and 11 S genotypes. The majority of snakes were multiply infected, with up to 4 distinct S and 11 distinct L segment genotypes in individual animals. This S/L imbalance was typical: in all cases intrahost L segment genotypes outnumbered S genotypes, and a particular S segment genotype dominated in individual animals and at a population level. We corroborated sequencing results by qRT-PCR and virus isolation, and isolates replicated as ensembles in culture. Numerous instances of recombination and reassortment were detected, including recombinant segments with unusual organizations featuring 2 intergenic regions and superfluous content, which were capable of stable replication and transmission despite their atypical structures. Overall, this represents intrahost diversity of an extent and form that goes well beyond what has been observed for arenaviruses or for viruses in general. This diversity can be plausibly attributed to the captive intermingling of sub-clinically infected wild-caught snakes. Thus, beyond providing a unique opportunity to study arenavirus evolution and adaptation, these findings allow the investigation of unintended anthropogenic impacts on viral ecology, diversity, and disease potential.     

Genetic exchange by recombination or reassortment is infrequent in natural populations of a tripartite RNA plant virus.

Two hundred seventeen field isolates of cucumber mosaic cucumovirus (CMV), sampled from 11 natural populations, were typed by RNase protection assay (RPA) using probes from the genomic RNAs of strains in subgroup I and in subgroup II of CMV strains. Most (85%) of the analyzed isolates belonged to subgroup I. For these subgroup I isolates, only two clearly different RPA patterns, A and B, were found for each of four probes representing RNA1, RNA2, and each of the two open reading frames in RNA3. On the basis of these RPA patterns for each probe, different haplotypes were defined. The frequency composition for these haplotypes differed for the various analyzed populations, with no correlation with place or year of sampling. This genetic structure corresponds to a metapopulation with local extinctions and recolonizations. Most subgroup I isolates (73%) belonged to haplotypes with RPA pattern A (type 1) or B (type 2) for all four probes. A significant fraction of subgroup I isolates (16%) gave evidence of mixed infections with these two main types, from which genetic exchange could occur. Genetic exchange by segment reassortment was seen to occur: the fraction of reassortant isolates was 4%, reassortment did not occur at random, and reassortants did not become established in the population. Thus, there is evidence of selection against reassortment between types 1 and 2 of subgroup I isolates. Aphid transmission experiments with plants doubly infected with type 1 and type 2 isolates gave further evidence that reassortment is selected against in CMV. Genetic exchange by recombination was detected for RNA3, for which two RPA probes were used. Recombinant isolates amounted to 7% and also did not become established in CMV populations. Sequence analyses of regions of RNA1, RNA2, and RNA3 showed that there are strong constraints to maintain the encoded sequence and also gave evidence that these constraints may have been different during divergence of types 1 and 2 and, later on, during diversification of these two types. Constraints to the evolution of encoded proteins may be related to selection against genetic exchange. Our data, thus, do not favor current hypotheses that explain the evolution of multipartite viral genomes to promote genetic exchange.     

The evolutionary genetics and emergence of avian influenza viruses in wild birds

We surveyed the genetic diversity among avian influenza virus (AIV) in wild birds, comprising 167 complete viral genomes from 14 bird species sampled in four locations across the United States. These isolates represented 29 type A influenza virus hemagglutinin (HA) and neuraminidase (NA) subtype combinations, with up to 26% of isolates showing evidence of mixed subtype infection. Through a phylogenetic analysis of the largest data set of AIV genomes compiled to date, we were able to document a remarkably high rate of genome reassortment, with no clear pattern of gene segment association and occasional inter-hemisphere gene segment migration and reassortment. From this, we propose that AIV in wild birds forms transient "genome constellations," continually reshuffled by reassortment, in contrast to the spread of a limited number of stable genome constellations that characterizes the evolution of mammalian-adapted influenza A viruses.     

Trials of direct detection and identification of Rhizoctonia solani AG 1 and AG 2 subgroups using specifically primed PCR analysis

Specifically primed polymerase chain reaction (PCR) analysis was used for direct detection and identification of Rhizoctonia solani isolates belonging to AG 1 subgroups (IA, IB, and IC) and AG 2 subgroups (2-1 and 2-2). A rapid DNA extraction method with a solution of sodium hydroxide was conducted to extract PCR templates. PCRspecific primer sets for each group were designed from sequences in the regions of the 28S ribosomal DNA of this fungus. The results of specifically primed PCR analysis showed that AG 1-IA, AG 1-IB, AG 1-IC, AG 2-1, and AG 2-2 primers sets contributed detection from the same AG isolates and could escape detection from different AG isolates at a high level of frequency. In this experiment, we suggested that our synthesized primer sets might provide a method for the direct detection and identification of AGs of R. solani.     

Role of Genetic Recombination in the Molecular Architecture of Papaya ringspot virus

Papaya ringspot virus (PRSV) has a single-stranded RNA genome and causes severe economic losses both in cucurbits and papaya worldwide. The extent to which the genome of PRSV is shaped by recombination provides an understanding of the molecular evolution of PRSV and helps in studying features such as host specificity, geographic distribution, and its emergence as new epidemics. The PRSV-P-Indian isolate was completely sequenced and compared with 14 other isolates reported from the rest of the world for their phylogenetic survey of recombination events. Cistron-by-cistron sequence comparison and phylogenetic analysis based on full-genome polyprotein showed two distinct groupings of Asian and American isolates, although PRSV-P and W-India clustered along with the American isolates. Recombination sites were found throughout the genomes, except in the small 6K1 protein gene. A significant proportion of recombination hotspots was found in the P1 gene, followed by P3, cylindrical inclusion (CI), and helper component proteinase (HcPro). Correlations between the presence of recombination sites, geographic distribution, and phylogenetic relationship provide an opportunity to establish the molecular evolution and geographic route of PRSV.     

Phylogenetic analysis of clinical herpes simplex virus type 1 isolates identified three genetic groups and recombinant viruses

Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen which establishes lifelong infections. In the present study, we determined the sequence diversity of the complete genes coding for glycoproteins G (gG), I (gI), and E (gE), comprising 2.3% of the HSV-1 genome and located within the unique short (US) region, for 28 clinical HSV-1 isolates inducing oral lesions, genital lesions, or encephalitis. Laboratory strains F and KOS321 were sequenced in parallel. Phylogenetic analysis, including analysis of laboratory strain 17 (GenBank), revealed that the sequences were separated into three genetic groups. The identification of different genogroups facilitated the detection of recombinant viruses by using specific nucleotide substitutions as recombination markers. Seven of the isolates and strain 17 displayed sequences consistent with intergenic recombination, and at least four isolates were intragenic recombinants. The observed frequency of recombination based on an analysis of a short stretch of the US region suggests that most full-length HSV-1 genomes consist of a mosaic of segments from different genetic groups. Polymorphic tandem repeat regions, consisting of two to eight blocks of 21 nucleotides in the gI gene and seven to eight repeats of 3 nucleotides in the gG gene, were also detected. Laboratory strain KOS321 displayed a frameshift mutation in the gI gene with a subsequent alteration of the deduced intracellular portion of the protein. The presence of polymorphic tandem repeat regions and the different genogroup identities can be used for molecular epidemiology studies and for further detection of recombination in the HSV-1 genome.     

Identification,genotyping, and molecular evolution analysis of duck circovirus

Duck circovirus (DuCV) is a contagious immunosuppressive virus affecting many duck species, which is responsible for multiple outbreaks in poultry industries worldwide. In this study, the first DuCV isolate GH01 was identified in Sichuan by PCR, which shared a high level of nucleotide identity (81.8–99.4%) with sequences of other DuCV isolates available in GenBank. Comparative phylogenetic and pairwise sequence comparison analyses indicated that DuCV could be divided into two genotypes (DuCV-1 and DuCV-2) and six subtypes (1a, 1b, 1c, 2a, 2b and 2c) based on the complete genome sequence. The results revealed that both DuCV-1 and DuCV-2 had evolved from the same ancestor but undergone divergent evolution. Interestingly, phylogenetic analyses indicated that three isolates were classified into a cluster DuCV-2a using complete DuCV genome sequence and cap gene, except rep gene. Recombination analyses revealed that DuCV-2a arose from recombination between DuCV-1a and DuCV-2b isolates within the rep genes, and the recombination events mainly occur both in non-structural protein coding region and structural protein coding region. In addition, the mechanisms of recombination supporting the genetic variability in DuCV isolates were investigated. Likewise, selective pressure indicated that purifying selection had been a major driving force in maintaining diversity among the DuCV isolates. Because eradicating the virus from commercial ducks is impossible, it is necessary to take effective control measures and implement them throughout the world.     

RubisCO Gene Clusters Found in a Metagenome Microarray from Acid Mine Drainage

The enzyme responsible for carbon dioxide fixation in the Calvin cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), is always detected as a phylogenetic marker to analyze the distribution and activity of autotrophic bacteria. However, such an approach provides no indication as to the significance of genomic content and organization. Horizontal transfers of RubisCO genes occurring in eubacteria and plastids may seriously affect the credibility of this approach. Here, we presented a new method to analyze the diversity and genomic content of RubisCO genes in acid mine drainage (AMD). A metagenome microarray containing 7,776 large-insertion fosmids was constructed to quickly screen genome fragments containing RubisCO form I large-subunit genes (cbbL). Forty-six cbbL-containing fosmids were detected, and six fosmids were fully sequenced. To evaluate the reliability of the metagenome microarray and understand the microbial community in AMD, the diversities of cbbL and the 16S rRNA gene were analyzed. Fosmid sequences revealed that the form I RubisCO gene cluster could be subdivided into form IA and IB RubisCO gene clusters in AMD, because of significant divergences in molecular phylogenetics and conservative genomic organization. Interestingly, the form I RubisCO gene cluster coexisted with the form II RubisCO gene cluster in one fosmid genomic fragment. Phylogenetic analyses revealed that horizontal transfers of RubisCO genes may occur widely in AMD, which makes the evolutionary history of RubisCO difficult to reconcile with organismal phylogeny.     

Frequency and dynamics of recombination within different species of human enteroviruses

Enteroviruses are members of the family Picornaviridae that cause widespread infections in human and other mammalian populations. Enteroviruses are genetically and antigenically highly variable, and recombination within and between serotypes contributes to their genetic diversity. To investigate the dynamics of the recombination process, sequence phylogenies between three regions of the genome (VP4, VP1, and 3Dpol) were compared among species A and B enterovirus variants detected in a human population-based survey in Scotland between 2000 and 2001, along with contemporary virus isolates collected in the same geographical region. This analysis used novel bioinformatic methods to quantify phylogenetic compatibility and correlations with serotype assignments of evolutionary trees constructed for different regions of the enterovirus genome. Species B enteroviruses showed much more frequent, time-correlated recombination events than those found for species A, despite the equivalence in population sampling, concordant with a linkage analysis of previously characterized enterovirus sequences obtained over longer collection periods. An analysis of recombination among complete genome sequences by computation of a phylogenetic compatibility matrix (PCM) demonstrated sharply defined boundaries between the VP2/VP3/VP1 block and sequences to either side in phylogenetic compatibility. The PCM also revealed equivalent or frequently greater degrees of incompatibility between different parts within the nonstructural region (2A-3D), indicating the occurrence of extensive recombination events in the past evolution of this part of the genome. Together, these findings provide new insights into the dynamics of species A and B enterovirus recombination and evolution and into the contribution of structured sampling to documenting reservoirs, emergence, and spread of novel recombinant forms in human populations.     

Differentiation of Bulgarian Isolates from Cucumber Mosaic Virus by Serological Methods

Cucumber mosaic virus (CMV) is of great importance to the Bulgarian economy and hence a detailed knowledge of its diversity under local geographic and climatic conditions is required. An extended study was carried out on CMV strains the currently occur in Bulgaria. Fifty-one isolates and strains found in different regions and various crops were biologically characterized and serologically differentiated into subgroups I and II using different variants of enzyme-linked immunosorbent assay (ELISA) [double antibody sandwich (DAS)-, antigen-coated plate (ACP)-, triple antibody sandwich (TAS)- with poly and monoclonal antibodies] and immunodiffusion tests. The ELISA modifications with monoclonal antibodies individually (ACP) or in combination with polyclonal antibodies (TAS-ELISA) are suitable for mass screening of CMV isolates. The hyperimmune sera against strains from CMV subgroups I and II were very efficient for use in isolate differentiation via gel double immunodiffusion. The results obtained correlated with the polymerase chain reaction and restriction fragment length polymorphism data reported by other authors. The majority of the isolates belonged to subgroup I, whereas 10, mainly from tomato and pepper, belonged to subgroup II. Most of the subgroup II isolates came from the north of Bulgaria. The results of the present study will help to clarify the virus epidemiology and to develop specific control measures.     

Molecular Evidence for Recombination in <Emphasis Type="Italic">Prunus</Emphasis> Necrotic Ringspot Virus

Genetic RNA recombination plays an important role in viral evolution. The evolutionary history of Prunus necrotic ringspot virus (PNRSV) has been extensively studied, but knowledge of recombination in its genome is still lacking. To investigate the recombination events in this virus, 67 accessions composed by 62 isolates retrieved from the databanks and five Tunisian isolates described in this study were analyzed. The use of RECCO algorithm which is based on cost minimization allowed us to detect several breakpoints in the coat protein gene (CP) of three out of five isolates from Tunisia and one from Poland. Moreover, a recombination signal was also detected in the putative cell-to-cell movement protein-encoding gene of an isolate from the USA. Tajima Neutrality test implemented in MEGA4 program indicated the occurrence of a high level of deletion/insertion events in the sequences. The evolutionary historical relationships were determined by constructing a dendrogram using neighbor joining, minimum evolution, maximum parsimony, maximum likelihood, and unweighted pair group method with arithmetic mean (UPGMA). The first four analyses gave similar results. Three classical groups (PE 5, PV 32, and PV 96) were delineated. The recombinant isolates from Tunisia clustered in a distinct clade except for one nonrecombinant (Ghernghezel) which revealed to be a member of PV 32 group. In contrast, UPGMA algorithm divided the Tunisian isolates in three distinct subgroups. Apart from recombination, reassortment is still an open question among many others and may also represent another way to explore the genetic diversity of PNRSV.     

The marsupial MHC: The Tammar Wallaby,Macropus eugenii,contains an expressed DNA-like gene on chromosome 1

Isolates of cauliflower mosaic virus (CaMV) differ in host range and symptomatology. Knowledge of their sequence relationships should assist in identifying nucleotide sequences responsible for isolate-specific characters. Complete nucleotide sequences of the DNAs of eight isolates of CaMV were aligned and the aligned sequences were used to analyze phylogenetic relationships by maximum likelihood, bootstrapped parsimony, and distance methods. Isolates found in North America clustered separately from those isolated from other parts of the world. Additional isolates, for which partial sequences were available, were incorporated into phylogenetic analysis of the sequences of genome segments corresponding to individual protein coding regions or the large intergenic region of CaMV DNA. The analysis revealed several instances where the position of an isolate on a tree for one coding region did not agree with the position of the isolate on the tree for the complete genome or with its position on trees for other coding regions. Examination of the distribution of shared residue types of phylogenetically informative positions in anomalous regions suggested that most of the anomalies were due to recombination events during the evolution of the isolates. Application of an algorithm that searches for segments of significant length that are identical between pairs of isolates or contain a significantly high concentration of polymorphisms suggested two additional recombination events between progenitors of the isolates studied and an event between the XinJing isolate and a CaMV not represented in the data set. An earlier phylogenetic origin for CaMV than for carnation etched ring virus, the caulimovirus used as outgroup in these analyses, was deduced from the position of the outgroup with North American isolates in some trees, but with non-North American isolates in other trees. Correspondence to: U. Melcher